Genetic separation of FK506 susceptibility and drug transport in the yeast Pdr5 ATP-binding cassette multidrug resistance transporter.
Overexpression of the yeast Pdr5 ATP-binding cassette transporter leads to pleiotropic drug resistance to a variety of structurally unrelated cytotoxic compounds. To identify Pdr5 residues involved in substrate recognition and/or drug transport, we used a combination of random in vitro mutagenesis and phenotypic screening to isolate novel mutant Pdr5 transporters with altered substrate specificity. A plasmid library containing randomly mutagenized PDR5 genes was transformed into appropriate drug-sensitive yeast cells followed by phenotypic selection of Pdr5 mutants. Selected mutant Pdr5 transporters were analyzed with respect to their expression levels, subcellular localization, drug resistance profiles to cycloheximide, rhodamines, antifungal azoles, steroids, and sensitivity to the inhibitor FK506. DNA sequencing of six PDR5 mutant genes identified amino acids important for substrate recognition, drug transport, and specific inhibition of the Pdr5 transporter. Mutations were found in each nucleotide-binding domain, the transmembrane domain 10, and, most surprisingly, even in predicted extracellular hydrophilic loops. At least some point mutations identified appear to influence folding of Pdr5, suggesting that the folded structure is a major substrate specificity determinant. Surprisingly, a S1360F exchange in transmembrane domain 10 not only caused limited substrate specificity, but also abolished Pdr5 susceptibility to inhibition by the immunosuppressant FK506. This is the first report of a mutation in a yeast ATP-binding cassette transporter that allows for the functional separation of substrate transport and inhibitor susceptibility.
Egner
R
R
Department of Molecular Genetics, University and Biocenter of Vienna, A-1030 Vienna, Austria.
Rosenthal
F E
FE
Kralli
A
A
Sanglard
D
D
Kuchler
K
K
D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000935:Antifungal Agents / Q000494:pharmacology; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology; D002462:Cell Membrane / Q000737:chemistry; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology; D003907:Dexamethasone / Q000378:metabolism / Q000494:pharmacology; D004352:Drug Resistance, Microbial / Q000235:genetics*; D018432:Drug Resistance, Multiple / Q000235:genetics*; D004958:Estradiol / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006360:Heat-Shock Proteins / Q000235:genetics / Q000502:physiology; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D020112:Rhodamine 123; D012235:Rhodamines / Q000378:metabolism / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D013379:Substrate Specificity; D016559:Tacrolimus / Q000494:pharmacology*; D022021:Tacrolimus Binding Proteins
D018528:ATP-Binding Cassette Transporters; D000935:Antifungal Agents; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; C092438:PDR5 protein, S cerevisiae; D012235:Rhodamines; D029701:Saccharomyces cerevisiae Proteins; C026188:rhodamine 6G; D020112:Rhodamine 123; D004958:Estradiol; D003907:Dexamethasone; D003513:Cycloheximide; D022021:Tacrolimus Binding Proteins; D016559:Tacrolimus